The fermentative production of succinate from renewable feedstocks will become increasingly competitive as petroleum prices increase. Succinate can serve as a substrate for transformation into plastics, solvents, and other chemicals currently made from petroleum (Lee et al., 2004; Lee et al., 2005; McKinlay et al., 2007; Wendisch et al., 2006; Zeikus et al., 1999). Many bacteria have been described with the natural ability to produce succinate as a major fermentation product (U.S. Pat. No. 5,723,322; Table 1). However, complex processes, complex media and long incubation times are often required.
A variety of genetic approaches have previously been used to engineer Escherichia coli strains for succinate production with varying degrees of success (Table 1). In most studies, titers achieved were low and complex medium ingredients such as yeast extract or corn steep liquor were required. Strain NZN111 produced 108 mM succinate with a molar yield of 0.98 mol succinate per mol of metabolized glucose (Chatterjee et al., 2001; Millard et al., 1996; Stols and Donnelly, 1997). This strain was engineered by inactivating two genes (pflB encoding pyruvate-formatelyase and ldhA encoding lactate dehydrogenase), and over-expressing two E. coli genes, malate dehydrogenase (mdh) and phosphoenolpyruvate carboxylase (ppc), from multicopy plasmids. Strain HL27659k was engineered by mutating succinate dehydrogenase (sdhAB), phosphate acetyltransferase (pta), acetate kinase (ackA), pyruvate oxidase (poxB), glucose transporter (ptsG), and the isocitrate lyase repressor (iclR). This strain produced less than 100 mM succinate and required oxygen-limited fermentation conditions (Cox et al., 2006; Lin et al., 2005a, 2005b, 2005c; Yun et al., 2005). Analysis of metabolism in silico has been used to design gene knockouts to create a pathway in E. coli that is analogous to the native succinate pathway in Mannheimia succiniciproducens (Lee et al., 2005 and 2006). The resulting strain, however, produced very little succinate. Andersson et al. (2007) reported the highest levels of succinate production by an engineered E. coli (339 mM) containing only native genes.
Other researchers have pursued alternative approaches that express heterologous genes in E. coli. The Rhizobium eteloti pyruvate carboxylase (pyc) was over-expressed from a multicopy plasmid to direct carbon flow to succinate. (Gokarn et al., 2000; Vemuri et al., 2002a, 2002b). Strain SBS550MG was constructed by inactivating the isocitrate lyase repressor (iclR), adhE, ldhA, and ackA, and over-expressing the Bacillus subtilis citZ (citrate synthase) and R. etli pyc from a multi-copy plasmid (Sanchez et al., 2005a). With this strain, 160 mM succinate was produced from glucose with a molar yield of 1.6.
More complex processes have also been investigated for succinate production (Table 1). Many of these processes include an aerobic growth phase followed by an anaerobic production phase. The anaerobic phase is often supplied with carbon dioxide, hydrogen, or both (Andersson et al., 2007; Sanchez et al., 2005a and 2005b; Sanchez et al., 2006; U.S. Pat. No. 5,869,301; Vemuri et al., 2002a and 2002b). In a recent study with a native succinate producer, A. succiniciproducens, electrodialysis, sparging with CO2, cell recycle, and batch feeding were combined (Meynial-Salles et al., 2007).
The subject invention provides various forms of microorganisms, such as strains of E. coli, that produce succinate at high titers and yields in mineral salts media during simple, pH-controlled, batch fermentations without the need for heterologous genes or plasmids. During development, an intermediate strain was characterized that produced malate as the dominant product.